Infection by the human paramyxovirus parainfluenza virus (HPIV) causes major respiratory diseases, including croup, bronchiolitis and pneumonia, which lead to illness or death in millions of infants and young children worldwide. HPIV infects by fusing its envelope directly with the host cell membrane. During entry, the viral surface glycoproteins HN (receptor-binding protein; hemagglutinin-neuraminidase) and F (fusion protein) cooperate to mediate fusion upon receptor binding. However, until an incoming virus meets its target cell, the fusion machine is inactive. Each step in viral entry intimately reflects the natural host cell surface; to understand -- and to interfere with -- natural infection, we consider the fundamental mechanisms that affect virus-cell interplay in the host. The long-term goal of our laboratory is to understand HPIV entry at a level of detail that permits design of antiviral drugs that hijack the HN-F machinery, and to understand the mechanistic commonalities among the fusion machineries of paramyxoviruses. The Objective of This Application is to define the mechanisms of the paramyxovirus HN-F machinery that are relevant for infection in the human. Aim 1. Determine HN's role in preventing premature activation of F-mediated fusion. The HPIV3 HN-F fusion machinery must be activated only at the right place and time. We will determine whether HN protects F from being triggered prematurely, and whether this role is critical in the natural host, preventing production of inactive viral particle and abortive infection. Using full-length mutant HN proteins and recombinant chimeric HN proteins, we will identify the domain(s) of HN that retain F in its pre-triggered state, and define the mechanisms of HN's protective role. Aim 2. Identify the key interactions between native HN and F that result in timely activation of F. Using infectious virus and engineered liposomes, we will capture serial intermediate states of the HPIV3 fusion complex during activation, to identify the structural rearrangements in the HN-F complex in its native state during the fusion process. Using mutant and chimeric HN-F complexes, we will define the determinants for HN's activation of F, and for HN's role in shepherding F through a series of transient intermediates in an ongoing HN-F complex, in native viruses and in real time. Aim 3. The HN-F fusion machine is tailored to the natural host: Identify the essential features of this virus-host interplay during vral entry, and exploit them for design of novel antiviral targets. Circulating HPIV3 viruses must attain a balance of HN-F properties that determine clinical infection; this balance may be hijacked for an effective antiviral strategy. Human clinical HPIV3 isolates will be used to determine the balance of HN-F features that determine viability in vivo, and whether perturbing the HN-F fusion machinery affects infection in vivo. Overall, our studies will move the picture of paramyxovirus infection to a paradigm relevant for human infection, providing the scientific basis for the first treatments for HPIV.